Analysis of ChiA-produced chitin oligosaccharide hydrolysis products
Hydrolysis of (GlcNAc)n(n = 2–6) was conducted in a 100-Lreaction mixture containing 0.08 M ChiA in 50 mM Tris–HCl buffer(pH 8.0) and 1.5 mM substrate.
The reaction was incubated at 20◦C.After incubation, the hydrolysis products were immediately mixed1:1 with 70% (v/v) acetonitrile to stop the hydrolysis.
Subsequently,a 10-L portion of the sample was injected into a TSK-Gel Amide-80column (0.46 × 25 cm2inner diameter; Tosoh, Tokyo, Japan) on anAgilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA,USA).
The elution was performed with 70% acetonitrile at a flowrate of 0.7 mL/min at 25◦C. The chitin oligosaccharide substratesand hydrolytic products were detected by absorbance at 210 nm.